1. Field of the Invention
The present invention concerns a method for the treatment of amyloidosis associated with Alzheimer's disease by administering to a patient an effective amount of at least one agent that modulates or affects the phosphorylation of proteins in mammalian cells.
2. Background Information
Alzheimer's disease (AD) is a brain disorder characterized by altered protein catabolism. From the work of several laboratories, altered protein phosphorylation has been implicated in the formation of the intracellular neurofibrillary tangles found in Alzheimer's disease. However, a role for protein phosphorylation in the catabolism of the amyloid precursor protein (APP), from which is derived the major component of amyloid plaques found in AD, has not been demonstrated.
Alzheimer's disease is the most common single cause of dementia in late life. Individuals with Alzheimer's disease are characterized by progressive memory impairments, loss of language and visuospatial skills and behavior deficits (McKhann et al., 1986, Neurology, 34:939-944). The cognitive impairment of individuals with Alzheimer's disease is the result of degeneration of neuronal cells located in the cerebral cortex, hippocampus, basal forebrain and other brain regions. Histologic analyses of Alzheimer's disease brains obtained at autopsy demonstrated the presence of neurofibrillary tangles (NFT) in perikarya and axons of degenerating neurons, extracellular neuritic (senile) plaques, and amyloid plaques inside and around some blood vessels of affected brain regions. Neurofibrillary tangles are abnormal filamentous structures containing fibers (about 10 nm in diameter) that are paired in a helical fashion, therefore also called paired helical filaments. Neuritic plaques are located at degenerating nerve terminals (both axonal and dendritic), and contain a core compound of amyloid protein fibers.
During the past several years, primary pathological markers associated with Alzheimer's disease have been characterized. The biochemical analyses of three forms of Alzheimer brain lesions, tangles, neuritic plaques, and cerebrovascular plaques, have revealed protein sequence information, and have facilitated subsequent cDNA cloning and chromosomal mapping of some of the corresponding genes. Immunological studies have identified several candidates for protein constituents of the paired helical filaments (PHF), including microtubule-associated protein 2 (MAP-2), tau, and ubiquitin.
A central feature of the pathology of Alzheimer's disease is the deposition of amyloid protein within plaques. The 4 kDa amyloid protein (also referred to as A4, APC, .beta.-amyloid or B/A.sub.4 peptide), is a truncated form of the larger amyloid precursor protein (APP) which is encoded by a gene localized on chromosome 21. Genetic analysis has revealed that the APP gene does cosegregate with familial Alzheimer's disease in certain families.
Initial studies of individuals with Down's syndrome (DS), caused by trisomy of chromosome 21, indicate that these individuals develop Alzheimer-like pathology beyond the second decade of life.
Age, genetic elements, and, possibly environmental factors appear to contribute to cellular pathology of Alzheimer's disease. A fundamental but unanswered question in the pathogenesis of Alzheimer's disease is the relationship between abnormalities of neurons and the deposition of amyloid. Specifically, the cellular origin of pathological events leading to the deposition of amyloid fibrils adjacent to some areas of the blood-brain barrier (cerebrovascular amyloid) and in the proximity of nerve terminals (neuritic plaques) in specific brain regions as well as extracellular amyloid in plaques cores is not known. Glenner and Wong have described the purification and characterization of meningeal amyloid from brains of both individuals with Alzheimer's disease (Glenner and Wong, 1984, Biochem. Biophys. Res. Commun., 120:885-890) or Down's Syndrome (Glenner and Wong, 1984, Biochem. Biophys, Res. Commun., 122:1131-1135) and determined the N-terminal peptide sequences. Among 24 residues analyzed, the two amyloid peptides showed only one difference, namely at amino acid position 11 (glutamine in Alzheimer's disease amyloid versus glutamic acid in Down's Syndrome amyloid) among 24 residues analyzed. Subsequent studies of amyloid from Alzheimer brain plaque cores revealed amino acid sequences identical to the reported Down's Syndrome cerebrovascular amyloid data (Masters et al, 1985, Proc. Natl. Acad. Sci. U.S.A., 82:4245-4249). cDNA analysis of APP transcripts from both normal tissue and Alzheimer brain material demonstrated the presence of the codon for glutamic acid at this position.
The availability of protein sequence information from the amyloid protein in Alzheimer brains enable the design of synthetic oligonucleotides complementary to the putative messenger RNA transcripts. Four groups have independently reported successful cloning of cDNAs including the region of the amyloid protein sequence. One group cloned the apparent full-length transcript (approximately 3.4 kb) for APP from a human fetal brain CDNA library. The 695-residue amyloid precursor protein (APP.sub.695) shows typical features of a glycosylated cell-surface transmembrane protein. The C-terminal 12 to 14 residues of the A4 protein reside in the putative transmembrane domain of the precursor and 28 N-terminal residues are in the "extracellular domain". Genomic mapping localized the APP gene on human chromosome 21 using human-rodent somatic cell hybrids. Applying in situ hybridization techniques, this gene was sublocalized to chromosome 21q21 and more recently at the border of 21q21-22.
Chromosome 21 has been the subject of intensive studies because of its involvement in Down's Syndrome (trisomy 21). While 95% of individuals with Down's Syndrome are trisomic for the entire chromosome 21, 2-3% are mosaics, i.e., trisomic in only some cells, and 3-4% are caused by triplication (translocation) of the distal part of the long arm (21q22) of chromosome 21. The occurrence of such translocation has led to the conclusion that Down's Syndrome can be attributed to trisomy of the distal part. (the "pathological region") of chromosome 21. To date, it is not known precisely where the breakpoint on the q arm of chromosome 21 is located, and it is not known whether individuals with Down's Syndrome, who have partial trisomy, develop Alzheimer pathology. In this context, it will be of particular interest to determine if the APP gene maps within the "pathological region" of chromosome 21. The localization of the APP gene on the long arm of chromosome 21, together with the apparent development of Alzheimer's disease pathology in individuals with Down's Syndrome, provides a potential mechanism for the formation of amyloid on the basis of over-expression of a number of genes on chromosome 21, including the APP gene. Initial studies of genomic DNA from sporadic (nonfamilial) Alzheimer's disease cases and "karyotypically normal" individuals with Down's Syndrome have implicated the presence of microduplication of a segment of chromosome 21 including the APP gene. However, subsequent analyses of large numbers of individuals with Alzheimer's disease by several laboratories has not confirmed these findings (Tanzi et al., 1987, Science, 238:666-669; St. George-Hyslop et al., 1987, Science, 238:664-666; Podlisny et al., 1987, Science, 238:669-671; Warren et al., 1987, Genomics, 1:307-3 12).
Chromosomal mapping experiments, using human APP probes in human/rodent cell hybrids, have shown cross-hybridization with mouse and hamster genomic DNA. Southern blot analysis of DNA from various species has indicated that the APP gene is highly conserved during evolution. Comparison of the mouse APP sequence with the sequence from rat shows 99% homology on the protein level; furthermore, the human sequence is 96.8% homologous to the mouse sequence and 97.3% homologous to the rat sequence. Based on the striking conservation of APP protein, Yamada et al. (1987, Biochem. Biophys. Res. Commun. 158:906-912) have calculated the evolutionary rate of changes at the amino acid level to be 0.1.times.10.sup.-9 /site/year, which is comparable to that of cytochrome C. Recently, K. White and colleagues have cloned a Drosophila gene which is highly homologous to large regions of the APP sequence. Northern blot experiments have confirmed these data at the level of mRNA and have demonstrated for various mammalian species the ubiquitous expression of APP transcripts in a number of different tissues (Manning et al., 1988, Brain Res., 427:293-297).
Kang et al. (Nature 325:733-736) reported the presence of two distinct bands (-3.2 kb and -3.4 kb) by Northern analysis of human fetal brain mRNA using APP cDNA as a probe. This finding suggests either differential splicing of mRNA or alternative usage of polyadenylation sites. Both posttranscriptional events were found to be operative following detailed investigation by several groups. First, Kang et al., supra, indicated a potential polyadenylation signal (AATAAA tandem repeat) 259 bp upstream of the 3' end of the reported APP full-length APP cDNA. The analysis of eight other full-length APP cDNA clones obtained from a human fetal brain cDNA library demonstrated in a 1:1 ratio between shorter cDNAs (-3.2 kb) using the first polyadenylation signal versus the original cDNA forms (-3.4 kb) using the second polyadenylation signal. Interestingly, all eight clones encoded for 695 residues of APP. The alternative use of different polyadenylation signals in APP transcripts was confirmed by other laboratories. A number of groups have screened several tumor cell-line derived cDNA libraries for the presence of APP transcripts and identified clones encoding new APP molecules containing an additional domain. This domain possesses striking homology to the Kunitz family of serine protease inhibitors. In particular, these cDNA sequences contain an additional 167 bp insert at residue 289 of the APP-695 precursor which encodes a 56 amino acid sequence of high sequence of homology to aprotinin, a well-characterized inhibitor of "trypsin-like" serine proteases. The peptide sequences flanking this region of insert are identical to the original APP.sub.695, clone resulting in an open reading frame of 751 residues (APP.sub.751). A third APP form has been isolated with another addition of a 19 amino acid domain at the C-terminal end of the 56 amino acid "aprotinin-like" region of APP.sub.751, thus resulting in a larger protein of 770 residues (APP.sub.770). Transient expression of APP.sub.770 in COS-1 cells conferred a marked inhibition of trypsin activity in cell lysates. Both additional domains have been found to be encoded by discrete exons and all three transcripts (APP.sub.695, APP.sub.751, APP.sub.770) are generated by differential splicing of a single gene on chromosome 21. These protease inhibitor domains have also recently been found to be present in mouse and rat species.
The relationship between the three different amyloid precursor forms and the formation of amyloid in Alzheimer's disease is not known. In particular, it is not known whether a specific form of APP contributes to A4 deposition. It is possible that either an imbalance in the relative expression levels of the three APP forms or their over-expression might be involved in Alzheimer's disease pathology. Initial in situ hybridization analyses using APP cDNA probes in human CNS sections indicated that many neuronal cell types express these mRNAs, but because of the nature of the probes used, these studies did not allow a differential analysis of the various APP transcripts. Furthermore, there is little documented correlation between APP mRNA levels, amyloid deposition and neuronal degeneration in Alzheimer's disease. However, it appears that high levels of APP mRNAs alone do not form a sufficient prerequisite or cellular pathology in either the aging or Alzheimer's disease brain (Higgins et al., supra). Specific probes which discriminate between the APP transcripts have been used for Northern analysis and the results suggest a developmental and tissue-specific pattern of expression of these mRNAs.
Recently, 5'-end cDNA probes from full-length APP cDNA clones have been used to isolate genomic clones containing the 5'-end of the APP gene, also referred to as precursor of Alzheimer's Disease A4 amyloid protein (PAD) gene (1988, EMBO J., 1:2807-2813; La Fauci et al, 1989, Biochem. Biophys. Res. Commun., 159:297-304). Approximately 3.7 kb of sequences upstream of the strongest RNA start site have been analyzed by Salbaum et al, 1988, supra. By a combination of primer extension and S1 protection analyses, five putative transcription initiation sites have been determined within a 10 bp region. This -3.7 kb region lacks a typical TATA box and displays a 72% GC-rich content in a region (-1 to -400) that confers promoter activity to a reporter gene in an in vivo assay system The absence of a typical TATA and CAAT box and the presence of multiple RNA start sites is suggestive of its function as a housekeeping gene but does not imply constitutive gene expression. The regulatory region contained within 400 bp upstream of the strongest RNA start site shows a variety of typical promoter-binding elements, including two AP-1 consensus sites, a single heat shock recognition consensus element, and several copies of a 9 bp-long GC-rich consensus sequence where sequence- specific binding has been shown to occur by gel-retardation studies. In addition, the CpG:GpC ratio in this promoter region has been found to be 1:1 in contrast to a 1:5 ratio found in many eukaryotic DNAs; CpG dinucleotides are known to control gene expression via DNA methylation. In addition, palindromic sequences capable of forming hairpin-like structures are found around the RNA start sites.
Recently, several groups of investigators have determined the consensus binding sequence (AT rich decamer) for a number of different homeobox proteins, which act most likely as transcription factors in specific regions during embryogenesis. As yet, target genes, which might be developmentally regulated by the homeobox proteins have not been identified. Such genes, however, will have an important role during embryogenesis and potentially throughout the entire lifespan. The APP gene promoter contains at least five homeobox binding sites upstream of the kNA start sites. Preliminary experiments have shown that the homeobox protein Hox-1.3 can bind at two of these sites. Thus, the APP gene, whose expression is developmentally regulated, appears to be a candidate gene for homeobox protein regulation. It is not known whether any of these putative recognition consensus elements translate the expression of the APP gene promoter.
Despite all that is known about the APP gene, the secondary defect leading to Alzheimer's disease is not yet known. With the exception of aged primates (Price et al., Bio Assays, 1989, 10:69-74), no laboratory animal model for Alzheimer's disease exists. The introduction of genes into the germline of animals is an extremely powerful technique for the generation of disease models which will lead to a better understanding of disease mechanisms including the mechanisms of Alzheimer's disease. Cell culture and in vitro systems cannot duplicate the complex physiological interactions inherent in animal systems. Transgenic animals have been successfully generated from a number of species including mice, sheep and pig. The gene or genes of interest are microinjected directly into the pronuclei of a one-cell embryo. A high percentage of reimplanted embryos develop normally and, in a significant proportion of progeny, the transgene becomes integrated into the chromosomal DNA. Usually, multiple copies of the transgene integrate as a head-to-tail array. Although mosaic animals can be generated, germline transmission of the transgene usually occurs.
In summary, Alzheimer's disease is characterized by certain neuropathological features including intracellular neurofibrillary tangles, primarily composed of cytoskeletal proteins, and extracellular parenchymal and cerebrovascular amyloid.